Deep UV Laser-Induced Fluorescence Detection of Unlabeled Drugs and Proteins in Microchip Electrophoresis

Abstract

Deep UV fluorescence detection at 266-nm excitation wavelength has been realized for sensitive detection in microchip electrophoresis. For this purpose, an epifluorescence setup was developed enabling the coupling of a deep UV laser into a commercial fluorescence microscope. Deep UV laser excitation utilizing a frequency quadrupled pulsed laser operating at 266 nm shows an impressive performance for native fluorescence detection of various compounds in fused-silica microfluidic devices. Aromatic low molecular weight compounds such as serotonin, propranolol, a diol, and tryptophan could be detected at low-micromolar concentrations. Deep UV fluorescence detection was also successfully employed for the detection of unlabeled basic proteins. For this purpose, fused-silica chips dynamically coated with hydroxypropylmethyl cellulose were employed to suppress analyte adsorption. Utilizing fused-silica chips permanently coated with poly(vinyl alcohol), it was also possible to separate and detect egg white chicken proteins. These data show that deep UV fluorescence detection significantly widens the application range of fluorescence detection in chip-based analysis techniques.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

“…16 However, the addition of fluorescent labels to proteins can affect their ligand binding, solubility and stability, and it is also difficult to ensure the attachment of only a single label per protein molecule. 17,18 The measurement of intrinsic protein fluorescence has also been demonstrated previously in microchannels for the label-free detection of proteins and their conformational changes, by using lasers [19][20][21] or light-emitting diodes (LED's) 22,23 for the excitation of samples. However, these techniques have not yet been sufficiently accurate to obtain thermodynamic parameters such as the free-energy of protein unfolding, as is required for use in drug-discovery, formulation or protein engineering applications.…”

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

“…With solid state lasers, nM detection limits of peptides [17] and proteins [18,19], as well as a pM detection limit for carbonic anhydrase [19] were reported. In contrast to these conventional CE methods protein separation with native UV detection on a quartz microchip with mM detection limit has only been demonstrated in the very recent past [20]. CE could thus be a method of choice for label-free single cell analysis.…”

Capillary electrophoresis of proteins 2003–2005

“…Deep UV LIF detection at 266 nm has been used to detect proteins separated by CZE on a chip [98]. The utility of the system has been proved by CZE of proteins from egg white.…”

“…A number of CE steps and components that are used in fused-silica capillaries have been transferred and successfully tested on a microchip including preconcentration on porous silica membrane [22,23], automated derivatization [29], microreactor with immobilized proteases and other proteins [181,182], UV detector [183,184], chemiluminescence detector [34,185], multiwavelength LIF detector [186], UV LIF detector [98], wall coatings [82,85], affinity CE [129], CIEF [80], and sheathless electrospray for MS [113,114].…”